Menaquinol Compositions and Methods of Treatment

ABSTRACT

The present application discloses methods for the efficient preparation of high purity compounds of the Formula I, and their methods of use.

RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application No.62/730,149 filed on Sep. 12, 2018.

FIELD OF INVENTION

The present invention relates to compounds, compositions andformulations, and combinations thereof, for the treatment of diseasesassociated with vitamin K, its reduced and bioactive form menaquinol,including osteoporosis and osteopenia.

BACKGROUND OF THE INVENTION

Vitamin K is known as a group of structurally similar, fat-solublevitamins. Vitamin K₂ or menaquinone has nine related compounds that canbe subdivided into the short-chain menaquinones (such as menaquinone-4or MK-4) and the long-chain menaquinones, such as MK-7, MK-8 andMK-9-12. The vitamins include phylloquinone (K1), menaquinones (K2) andmenadione (K3). Plants synthesize vitamin K1 while bacteria can producea range of vitamin K2 forms, including the conversion of K1 to K2 bybacteria in the small intestines. Vitamin K3 is synthetic version of thevitamin, and due to its toxicity, has been banned in by the US Food andDrug Administration for human uses.

It has been established that taking broad-spectrum antibiotics canreduce vitamin K production in the gut by nearly 74% in people comparedto those not taking these antibiotics. Diets that are low in vitamin Kalso decrease the body's vitamin K concentration. Vitamin K1 ispreferentially used by the liver as a clotting factor. Vitamin K2 isused preferentially in the brain, vasculature, breasts and kidneys.Vitamin K2 contributes to production of myelin and sphingolipids (fatsessential for brain health) and protects against oxidative damage in thebrain. Vitamin K2, such as MK-4, promotes bone health by stimulatingconnective tissue production in bone.

Vitamin K2, which is the main storage form in animals, has severalsubtypes, which differ in chain length of the isoprenoid group orresidue in the side chains. These vitamin K2 homologues are calledmenaquinones, and are characterized by the number of isoprenoid residuesin their side chains. For example, MK-4 has four isoprene residues inits side chain, and is the most common type of vitamin K2 in animalproducts. MK-4 is normally synthesized from vitamin K₁ in certain animaltissues (arterial walls, pancreas and testes) by replacement of thephytyl group with an unsaturated geranyl group containing four isopreneunits. Unlike MK-4, MK-7 is not produced by human tissue. MK-7 may beconverted from phylloquinone K₁) in the colon by E. coli bacteria. MK-4and MK-7 are sold in the U.S. in dietary supplements for bone health.MK-4 has been shown to decrease the incidence of fractures. MK-4, at adose of 45 mg daily, has been approved by the Ministry of Health inJapan since 1995 for the prevention and treatment of osteoporosis.

Osteoporosis is a disease of bone that leads to an increased risk offracture. In osteoporosis the bone mineral density (BMD) is reduced,bone micro architecture is disrupted, and the amount and variety ofnon-collagenous proteins in bone is altered. The World HealthOrganization define osteoporosis (in women) as a bone mineral density2.5 standard deviations below peak bone mass, that is, for an average30-year-old healthy female. Osteoporosis is most common in women aftermenopause (referred to as postmenopausal osteoporosis). Osteoporosis mayalso develop in men, and may occur in anyone in the presence ofparticular hormonal disorders and other chronic diseases or as a resultof medications, specifically glucocorticoids, when the disease is calledsteroid- or glucocorticoid-induced osteoporosis and as a result ofnutritional deficiency states or other metabolic disorders, for example,hyponatremia or as a secondary consequence of cancer.

Osteopenia is a condition where bone mineral density is lower thannormal. It is considered by many doctors to be a precursor toosteoporosis. Specifically, osteopenia is defined as a bone mineraldensity T score between −1.0 and −2.5. Osteopenia can be induced underspecific conditions such as long-term bed rest.

The underlying mechanism in most cases of osteoporosis is an imbalancebetween bone resorption and bone formation. In normal bone, there isconstant matrix remodelling of bone. It has been established that up to10% of all bone mass may be undergoing remodelling at any point in time.Bone is resorbed by osteoclast cells, which are derived from bone marrowprecursor cells. In the remodelling process new bone is deposited byosteoblast cells. The three main mechanisms by which osteoporosisdevelops include an inadequate peak bone mass (the skeleton developsinsufficient mass and strength during growth), excessive bone resorptionand inadequate formation of new bone during remodelling. Hormonalfactors strongly determine the rate of bone resorption; lack of estrogen(e.g. as a result of menopause) increases bone resorption as well asdecreasing the deposition of new bone that normally takes place inweight-bearing bones. In addition to estrogen, calcium metabolism playsa significant role in bone turnover, and deficiency of calcium andvitamin D leads to impaired bone deposition; in addition, theparathyroid glands react to low calcium levels by secreting parathyroidhormone, which increases bone resorption to ensure sufficient calcium inthe blood. Medications used for the treatment of osteoporosis includescalcium, vitamin D, vitamin K, bisphosphonates, Calcitonin,Teriparatide, strontium ranelate, hormone replacement and selectiveestrogen receptor modulators.

It has been established that cardiovascular disease (CVD) is the mostfrequent cause of death in patients with chronic kidney disease (CKD).When compared to the general population, the cause of death attributedto CVD is about 10-20 times higher in CKD patients when they are beingtreated with hemodialysis. In addition, it has been demonstrated thatvascular calcification and the correlated arterial stiffness isprevalent in the incidence of CVD. In addition, patient with CKDundergoing dialysis treatment have a 3 times higher risk of bonefractures, such as vertebral fractures and other type of bone fractures.

Vitamin K, including MK-7, are present in low concentrations in atypical diet. It has also been established that there exists a directcorrelation between the level of vitamin K in a patient's blood and theincidence of vascular calcification, bone density and bone strength.Accordingly, the supplemental use of vitamin K, such as MK-7 and itsalso fat-soluble hydroquinone (menaquinol) derivatives as disclosedherein, may provide significant clinical benefit for reducing vascularcalcification noted, in part, by arterial stiffness, and increase bonemineralization or increase in bone mineral density, that will help treator prevent CVD, and treat or prevent bone diseases in patients with CKD.In one aspect, the disclosed method for the administration of MK-7 andits fat-soluble hydroquinone derivatives, or combinations thereof, maybe used in the treatment or reduction of vascular calcification,increase in bone mineral density and for the treatment, reduction orprevention of bone diseases, such as in patients with CKD.

It has also been established that in food products, vitamin K1 is boundto the chloroplast membrane of leafy green vegetables. MK-4, which isderived from the conversion of menadione, a synthetic analog of vitaminK, is found in animal products such as eggs and meats. Long chainmenaquinones such as MK-7, MK-8 and MK-9, are found in fermented foodssuch as cheese, curd and sauerkraut. It has also been established thatthe effects of long chain MK-n such as MK-7 on normal blood coagulationis greater and longer lasting than vitamin K1 and MK-4. MK-7 has alsobeen shown to have a long half-life in serum when compared to MK-4,providing a better carboxylation-grade of osteocalcin compared toVitamin K1. See Sato et al., Nutrition Journal, 2012, 11:93.

Nutritional doses of MK-7 can be established to be well absorbed inhumans, and as a consequence, provide a significant increase in theserum for MK-7 levels. However, very little information is known ofMK-7, and menaquinol-7, primarily because MK-7 and menaquinol-7, are notreadily available nor commercially accessible via standard syntheticmethods.

In one embodiment, the present application discloses a novel andefficient method for the preparation of menaquinol-7 and itshydroquinone derivatives. The novel method uses a nickel(0)-catalyzedcoupling reaction and uses readily available starting materials, andprovides menaquinol-7 and its hydroquinone derivatives in high yieldsand high chemical purity.

The foregoing examples of the related art and limitations are intendedto be illustrative and not exclusive. Other limitations of the relatedart will become apparent to those of skill in the art upon a reading ofthe specification and a study of the drawings or figures as providedherein.

SUMMARY OF THE INVENTION

Therefore, a continuing need exists for formulations that are effectivefor these indications. The following embodiments, aspects and variationsthereof are exemplary and illustrative are not intended to be limitingin scope.

In one aspect, there is provided a chemoselective method for preparing amenaquinol compound with high regioselectivity, high isomeric purity andhigh chemical purity, the method comprising:

(a) contacting a compound of the Formula III:

where n is 6, 7, 8 or 9;

with a trialkylaluminum compound of the formula R₃Al, together with a Zrcatalyst, in an aprotic solvent to form a mixture of compounds of theFormulae IVa and IVb:

wherein R is selected from the group consisting of CH₃— and CH₃CH₂—;

(b) contacting the intermediate compound of the Formulae IVa and IVbwith a chloromethylquinone of the Formula V and a transition metalcatalyst:

to provide a mixture comprising a compound of the Formulae VIa and theregioisomer VIb:

In one variation of the trialkylaluminum compound of the formula R₃Al, Ris —CH₃. In one aspect, the method further comprises crystallizing themixture of the Formulae VIa and VIb using a solvent mixture to form amixture of the compound of the Formula VIa and VIb, wherein the ratio ofVIa to VIb is greater than 99.8:0.2, and the purity of VIa is greaterthan 99.6% as determined by HPLC. In another aspect of the above method,the purity of VIa is greater than 99.8% as determined by HPLC. The levelof purity of the products achieved using the present synthesis providespharmaceutical grade compositions that are greater that those productsobtained by fermentation processes.

In one variation of the method in step (b), the unreacted or excesstrialkylaluminum compound and the aprotic solvent in the reaction arenot removed from the reaction mixture before contacting the intermediatecompound with a compound of Formula V. On a large scale process, theelimination of this processing step reduces the potentially hazardoushandling of the pyrophoric compound. In one variation, thetrialkylaluminum compound is Me₃Al or Al₂Me₆. In one variation, thezirconium catalyst is an organozirconium catalyst. In another variation,the organozirconium catalyst is selected from the group consisting ofzirconocene dichloride (Cp₂ZrCl₂), rac-(ebi)ZrCl₂ and rac-(ebthi)ZrCl₂.In another variation, the metal catalyst is (Ph₃P)₂Ni(0), or(Ph₃P)₂Pd(0), that may be prepared from the addition of NiCl₂ or PdCl₂(or other Ni(II) and Pd(II) salt precursors) to 2 PPh₃ followed byaddition of 2 equivalents of n-BuLi in an aprotic solvent such as THF.In another variation, the zirconium catalyst is added as a solution inan aprotic solvent such as toluene, CF₃Ph, DCM or 1,2-dichloroethane.

In another aspect of the method, the metal catalyst is a nickel(0)catalyst. In another aspect, the nickel(0) catalyst is (Ph₃P)₂Ni(0). Inanother aspect, more than 1 molar equivalent to 1.4 molar equivalents ofthe trialkylaluminum compound is added relative to the compound of theFormula III. In one variation, 1.2 to 1.4 molar equivalents of thetrialkylaluminum compound is added relative to the compound of theFormula III. In another variation, about 1.2 molar equivalents of thetrialkylaluminum compound is added relative to the compound of theFormula III.

In another aspect of the above method, the solvent mixture forcrystallization of the compound of the Formulae VIa comprises ethylacetate and a C₁-C₃ alcohol. In one variation, the C₁-C₃ alcohol isselected from methanol, ethanol, propanol and isopropanol. In anothervariation, the EtOAc:C₁-C₃ alcohol is from about 1:5 to about 2:5. Inanother variation, the solvent mixture is EtOAc:EtOH at a ratio of 1:5.In another variation, the solvent mixture comprises of about 15-20%EtOAc in EtOH, 20-25% EtOAc in EtOH, or about 20-22% EtOAc in EtOH. Inanother variation, the solvent mixture comprises of about 25-30% EtOAcin EtOH.

In another embodiment, there is provided a pharmaceutically puremenaquinone compound of the Formula VIa:

wherein n is 6, 7, 8, or 9; and wherein the regioisomer of VIa isgreater that 99.8% as measured by HPLC, and the chemical purity of VIais greater than 99.6% as measured by HPLC. In one variation of thecompound VIa, n is 7, and the regioisomer of VIa is greater that 99.8%by HPLC, and the chemical purity of VIa is greater than 99.6% asmeasured by HPLC. In another aspect of the above compound, thepharmaceutically pure menaquinone compound where n is 6.

In one variation, there is provided a pharmaceutically pure compound ofthe Formula VIa prepared by the method as disclosed above, wherein n is6, 7, 8 or 9; and wherein the regioisomer of VIa is greater that 99.8%as measured by HPLC, and the purity is greater than 99.6% as measured byHPLC.

In another aspect, the method further comprises contacting the compoundof the Formula VIa with a metal or other reducing agent and an acid orother source of protons for a sufficient period of time under conditionsto form a menaquinol compound of the Formula VII:

wherein n is 6, 7, 8 or 9.

In one variation, the metal is Zn, SnCl₂, FeCl₃, FeCl₃ 3 H₂O and FeCl₃ 6H₂O and the acid is acetic acid or hydrochloric acid. In anothervariation, the reduction may be performed with N,N-diethylhydroxylamine(DEH) in HCl, such as dilute HCl. In another variation, the reductionmay be performed with FeCl₃ or FeCl₃ 3 H₂O and an acid such as HCl. Inone variation of the method, the reduced product may be isolated orpurified, or the reduced product may be further acylated in situ,without any further isolation or purification.

In another aspect of the method, the compound of the Formula VII isacylated with an acylating agent, for purposes of stabilization andisolation, selected from the group consisting of:

a) an acid halide selected from the group consisting of Formulae 15.a,16.a, 17.a, 18.a, 20.a, 21.a, 22.a, 23.a, 24.a, 25.a, 26.a, 27.a and28.a, wherein X is —Cl, Br, —I, as well as any of several other knownleaving groups in the art (e.g., imidazolyl, —SO₂R and —SO₂Ar, etc.where Ar is phenyl, toluyl and substituted phenyl and R is methyl,ethyl, phenyl and substituted phenyl) and a base selected from the groupconsisting of Cs₂CO₃, CsHCO₃, CsOH, LiCO₃, Na₂CO₃, K₂CO₃, KHCO₃, NaOAc,NaHCO₃, or other organic bases (e.g., Et₃N, DABCO, DBU, DBN, etc.); or

b) an acid anhydride selected from the group consisting of Formulae15.a, 16.a, 17.a, 18.a, 20.a, 21.a, 22.a, 23.a, 24.a, 25.a, 26.a, 27.aand 28.a, wherein X is —OC(O)R′ wherein R′ is —C₁₋₆alkyl and Zn, or abase selected from the group consisting of Cs₂CO₃, CsHCO₃, CsOH, LiCO₃,Na₂CO₃, K₂CO₃, KHCO₃, NaOAc and NaHCO₃;

wherein: P¹ and P² are each independently a protecting group selectedfrom the group consisting of —CH₂C₆H₅, -THP (tetrahydropyranyl) or P¹and P² together with the oxygen to which they are attached form a cyclicacetonide, benzyl acetal, or p-methoxybenzyl acetal;

P³ is a hydroxyl protecting group selected from the group consisting of-THP, acetyl, benzoyl, β-methoxyethoxymethyl ether (MEM),dimethoxytrityl, methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB),methylthiomethyl ether, pivaloyl (Piv) and trityl (Tr); and

each R³ and R⁴ is independently H, —CH₃, —CH₂CH₃ and —CH₂C₆H₅;

to form a menaquinol compound of the Formula I

wherein:

m is 7, 8, 9 or 10; and

each R¹ and R² is independently H or selected from the group consistingof:

In one variation, the compound of the Formula VII is prepared andacylated in situ with the acylating agent. In another variation, theacylated products are the ester derivatives of statin compounds such asrosuvastatin, pitavastatin, atorvastatin, or esters derived from omega-3fatty acids such as alpha-linolenic acid, stearidonic acid,eicosapentaenoic acid and dodecahexenoic acid, or esters derived fromacids found in coffee, such as caffeic acid, ferulic acid, chlorogenicacid and quinic acid, as their hydroxyl protected derivatives. In onevariation, R′ is —CH₃ or —CH₂CH₃, isopropyl or t-butyl. In anothervariation of the acylation reaction, the acylation may be performed in asolvent selected from the group consisting of ethyl acetate, THF,toluene, diethyl ether, dichloromethane or acetonitrile.

In another aspect, the method comprises further removing the hydroxylprotecting group R¹, R², R³, R⁴, P¹, P² and P³. The removal of theprotecting group such as —CH₂C₆H₅ may be performed, for example, byhydrogenation with hydrogen in Pd/C. Protecting groups such as -THP(tetrahydropyranyl), acetyl, benzoyl, β-methoxyethoxymethyl ether (MEM),dimethoxytrityl, methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB),methylthiomethyl ether, pivaloyl (Piv) and trityl (Tr); a cyclicacetonide, acetals, ketals such as benzyl acetal or p-methoxy-benzylacetal may be removed by acid hydrolysis. Standard methods for thepreparation, formation and removal of hydroxyl protecting groups may befound, for example in T. W. Greene, Protecting Groups in OrganicSynthesis, 3rd edition, John Wiley & Sons, Inc. 1999.

In another embodiment, the application provides a menaquinol compound ofthe Formula I:

wherein:

m is 7, 8, 9 or 10;

each of R¹ and R² is independently H or is independently selected fromthe group consisting of:

In another aspect, the application discloses the compounds of theFormula I, wherein:

R¹ and R² are both the residue 15;

R¹ and R² are both the residue 16;

R¹ and R² are both the residue 17;

R¹ and R² are both the residue 18;

R¹ and R² are both the residue 20;

R¹ and R² are both the residue 21;

R¹ and R² are both the residue 22;

R¹ and R² are both the residue 23;

R¹ and R² are both the residue 24;

R¹ and R² are both the residue 25;

R¹ and R² are both the residue 26;

R¹ and R² are both the residue 27; and R¹ and R² are both the residue28.

In another aspect, the application discloses the compounds of theFormula I, wherein:

R¹ is H and R² is the residue 15; R² is H and R¹ is the residue 15;

R¹ is H and R² is the residue 16; R² is H and R¹ is the residue 16;

R¹ is H and R² is the residue 17; R² is H and R¹ is the residue 17;

R¹ is H and R² is the residue 18; R² is H and R¹ is the residue 18;

R¹ is H and R² is the residue 20; R² is H and R¹ is the residue 20;

R¹ is H and R² is the residue 21; R² is H and R¹ is the residue 21;

R¹ is H and R² is the residue 22; R² is H and R¹ is the residue 22;

R¹ is H and R² is the residue 23; R² is H and R¹ is the residue 23;

R¹ is H and R² is the residue 24; R² is H and R¹ is the residue 24;

R¹ is H and R² is the residue 25; R² is H and R¹ is the residue 25;

R¹ is H and R² is the residue 26; R² is H and R¹ is the residue 26;

R¹ is H and R² is the residue 27; R² is H and R¹ is the residue 27; and

R¹ is H and R² is the residue 28; R² is H and R¹ is the residue 28.

In another embodiment, there is provided a compound of the Formula I:

wherein: m is 7, 8, 9 or 10;

each of R¹ and R² is independently H or is independently selected fromthe group consisting of:

wherein each R³ and R⁴ is independently H, —CH₃, —CH₂CH₃ and —CH₂C₆H₅.

In one variation of the above compound, R³ and R⁴ are both —CH₃. Inanother variation, each R³ and R⁴ is independently H and —CH₂C₆H₅.

In another aspect of the disclosed method of treatment and method ofprevention as recited herein, the compound is of the Formula III:

wherein: m is 7, 8, 9 or 10; and each of R¹ and R² is independently H oris independently selected from the group consisting of —C(O)C₁₋₆alkyl.As used herein, the group C₁₋₆alkyl includes methyl, ethyl, propyl,iso-propyl, cyclopropyl, butyl, iso-butyl, pentyl, iso-pentyl, hexyl,cyclohexyl and 1-methylpentyl. In one variation, the compound is of theFormula IIIa, wherein m is 7 and both R¹ and R² are —C(O)C₁₋₆alkyl. Inanother variation, the compound is of the Formula IIIb, wherein m is 7and both R¹ and R² are —C(O)CH₃.

In another aspect, the application discloses a pharmaceuticalcomposition comprising a therapeutically effective amount of amenaquinol compound as disclosed above, or a mixture thereof, and apharmaceutically acceptable excipient, wherein the composition iseffective for the treatment of a condition associated with vitamin Kselected from for the treatment of osteoporosis and arteriosclerosis.

In another aspect, the present application discloses a method for thetreatment of a disease in a mammal selected from the group consisting ofneurodegenerative diseases, retinopathy, rheumatoid polyarthritis,atherosclerosis, amyotrophic lateral sclerosis, cerebral ischemia,cataracts, systemic infections, pathologies associated with cutaneousaging and with senescence in tissues, pathologies associated withmitochondrial dysfunction, cachexia associated with under nutrition,wherein the treatment is associated with the increase in the longevityof mammals, the method comprises the administration of a therapeuticallyeffective amount of a compound or composition comprising a menaquinolcompound as disclosed above, or a mixture thereof.

In another embodiment, there is provided a method for treating a mammalwith a disease selected from the group consisting of vitamin Kdeficiency, osteoporosis, a proliferative disease, and a cardiovasculardisease, comprising administering to the mammal a therapeuticallyeffective amount of a compound as disclosed herein, or a mixturethereof. In another aspect of the method, the proliferative disease isselected from the group consisting of cancer, leukemia and aninflammatory disease.

In another embodiment, there is provided a method for the treatment orprevention of osteoporosis and/or osteopenia, the method comprisingadministering to a patient in need of treatment, a therapeuticallyeffective amount of a composition comprising a compound as disclosedabove, or a mixture thereof.

In another embodiment, there is provided a method of treating,preventing, slowing the progression of, arresting, and/or reversingcalciphylaxis in a mammal in need thereof, the method comprisingadministering to the mammal a therapeutically effective amount of acomposition comprising substantially pure menaquinol compound asdisclosed herein, and a pharmaceutically acceptable excipient, toprevent, slow the progression of, arrest, or reverse calciphylaxis. Inone aspect of the method, the mammal has distal calciphylaxis and/orcentral calciphylaxis. In another aspect, the mammal has diabetes,chronic kidney disease or end stage renal disease. In another aspect,the mammal has stage 3, stage 4 or stage 5 chronic kidney disease. Inanother aspect of the method, the mammal is undergoing hemodialysis. Inyet another aspect, the mammal is receiving non-warfarin-basedanti-coagulant therapy.

In another aspect of the above method, the anti-coagulant therapy isoral anti-coagulation therapy. In another aspect, the anti-coagulationtherapy comprises an inhibitor of Factor Xa activity selected fromapixaban, rivaroxaban, betrixaban, edoxaban, otamixaban, letaxaban,eribaxaban or fondaparinux; or Factor IIa activity selected fromdabigratran or argatroban. In another aspect, the mammal has chronicobstructive pulmonary disease (COPD). In another aspect, the mammal hasa calciphylaxis-related dermal lesion. In another aspect of the method,administration of the composition reduces the total surface area of thedermal lesion by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or100%. In another aspect of the method, administration of thesubstantially pure compound as disclosed herein, to the mammal increasesthe mammal's serum T50 value by at least 10%, 20%, 30%, 40%, 50%, 60%,70%, 80%, 90% or 100%) relative to the mammal's serum T50 value prior toadministration of the disclosed compound. In another aspect,administration of the disclosed compound increases a ratio of acarboxylated to a non-carboxylated of a Vitamin K dependent protein inplasma of the mammal after administration of the composition is greaterthan prior to administration of the composition. In one aspect of themethod, the increase of the ratio of a carboxylated to anon-carboxylated of a Vitamin K dependent protein in plasma of themammal after administration of the composition is by at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95%, or 100% relative to the ratio prior to administration.

In certain embodiments of the above, the administration of the disclosedcompounds decreases the amount of a non-carboxylated Vitamin K-dependentprotein in the subject's plasma, for example, by at least 5%, 10%, 15%,20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95% or 100% relative to the amount prior to administration of thecompounds. In certain variations, the Vitamin K-dependent protein isselected from Matrix Gla Protein (MGP), Growth Arrest Specific Gene 6(Gas-6) protein, PIVKA-II protein, osteocalcin, activated Protein C,activated Protein S, factor II, factor VII, factor IX, and factor X.

In certain variation of the above methods, the administration of thecompounds increases the plasma level of osteoprotegerin or Fetuin A, forexample, by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% relative to theplasma concentration of osteoprotegerin or Fetuin A prior toadministration of the compounds. In other variations, the administrationof the compounds decreases the plasma level of D-Dimer or HighlySensitive C Reactive Peptide (hs-CRP), for example, by at least 5%, 10%,15%, 20%, 25%, 30%, 35%, 40%, 45% 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or 100% relative to the plasma concentration of D-Dimer orHighly Sensitive C Reactive Peptide (hs-CRP) prior to administration ofthe compounds.

In certain variations of the above methods, the method may includeadministering from about 2 mg to about 750 mg of the compound to thesubject per day. In other variations, the method may includeadministering from about 5 mg to about 750 mg of the compound to thesubject per day. In other variations, the method may includeadministering from about 2 mg to about 500 mg of the compound to thesubject per day. In other variations, the method may includeadministering from about 5 mg to about 500 mg of the compound to thesubject per day. In certain variations, the method can includeadministering from about 2 mg to about 250 mg of the compound to thesubject per day. In other variations, the method may includeadministering from about 5 mg to about 250 mg of the compound to thesubject per day. In other variations, the method may includeadministering from about 2 mg to about 100 mg of the compound to thesubject per day. In other variations, the method may includeadministering from about 5 mg to about 100 mg of the compound to thesubject per day. In other variations, the method may includeadministering from about 10 mg to about 75 mg of the compound to thesubject per day, for example, administering 10, 25, 50 or 75 mg of thecompound to the subject per day.

In certain variations, the compound is administered to the subject forat least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year,or indefinitely as needed. If the subject is undergoing hemodialysis,the compound may be administered to the subject for a period thatincludes at least the duration of hemodialysis.

In another variation of the method for treatment of calciphylaxis, inaddition to measuring the change/reduction in lesion size followingadministration of the disclosed compounds, pre and post drug dosingadministration, a biopsy may be taken of the relevant lesions using vonKassa Staining to determine tissue levels of PTH and evidence of changein calcium and phosphate deposition in dermal arterioles.

As disclosed herein, the presence of a uremic oxidative blockade isdetermined by measuring increased plasma lipid peroxidation, e.g., bydetection of increased F2 isoprostanes (Morrow et al. (1990) “A seriesof prostaglandin F2-like compounds are produced in vivo by humans by anon-cyclooxygenase, free radical-catalyzed mechanism,” PROC. NATL. ACAD.SCI. USA 87:9383-9387), increased isolevuglandin-plasma protein adducts(Salomon et al. (2000) “Isolevuglandin-protein adducts in humans:Products of free radical induced lipid oxidation through the isoprostanepathway,” BIOCHIM BIOPHYS ACTA 1485:225-235), increased breath ethane(Handelman et al. (2000) J AM. SOC. NEPHROL. 11:271A); increased proteinand amino acid oxidation, e.g., by detection of tyrosine residueoxidation (Heinecke et al. (1999) “Detecting oxidative modification ofbiomolecules with isotope dilution mass spectrometry: Sensitive andquantitative assays for oxidized amino acids in proteins and tissues,”METHODS ENZYMOL. 300:124-144), cysteine or methionine residue oxidation,lysine oxidation and threonine oxidation, thiol oxidation and carbonylformation in plasma proteins (Himmelfarb et al. (2000) “Plasma proteinthiol oxidation and carbonyl formation in chronic renal failure,” KIDNEYINT. 58:2571-2578); reactive aldehyde formation, e.g., by detectingglyoxal, methylglyoxal, acrolein, glycoaldehyde, and parahydroxyphenacetaldehyde (Miyata et al. (1999) “Alterations in nonenzymaticbiochemistry in uremia: Origin and significance of ‘carbonyl stress’ inlong-term uremic complications. KIDNEY INT. 55:389-399); increasedreactive carbonyl compounds, e.g., by measuring hydrazine formationafter reaction with 2,4-dinitrophenylhydrazine; diminished plasmaglutathione levels and glutathione peroxidase function (Ceballos-Picotet al. (1996) “Glutathione antioxidant system as a marker of oxidativestress in chronic renal failure,” FREE RADIC. BIOL. MED. 21:845-853);and increased ratio of oxidized to reduced thiols (Hultberg et al.(1995) “Reduced, free, and, total fractions of homocysteine and otherthiol compounds in plasma from patients with renal failure,” NEPHRON70:62-67; Himmelfarb et al. (2002) “Plasma aminothiol oxidation inchronic renal failure,” KIDNEY INT 61:705-716; Ward et al.“Polymorphonuclear leukocyte oxidative burst is enhanced in patientswith chronic renal insufficiency,” J AM. SOC. NEPHROL. 5:1697-1702).

In another embodiment, there is provided a method of treating,preventing, slowing the progression of, arresting and/or reversingtissue calcification in a pre-diabetic mammal (or subject) withdiabetes, chronic kidney disease or a combination thereof, and in needthereof, the method comprising administering to the mammal at least 2 mgof substantially pure compound as disclosed herein per day, to prevent,slow the progression of, and/or arrest tissue calcification, wherein thecompound is administered in a pharmaceutical composition. In anotheraspect of the method, the mammal has diabetes. In yet another aspect,the mammal has type II diabetes; or the mammal has been diagnosed aspre-diabetic. In another aspect, the mammal has chronic kidney disease.In another aspect of the above method, the mammal has stage 4 or 5chronic kidney disease/end stage renal disease. In yet another aspect,the mammal is undergoing hemodialysis. In another aspect, the mammal isreceiving non-warfarin based anti-coagulant therapy. In another aspect,the anti-coagulant therapy is oral anti-coagulation therapy. In anotheraspect of the method, the anti-coagulation therapy comprises aninhibitor of Factor Xa activity selected from apixaban, rivaroxaban,betrixaban, edoxaban, otamixaban, letaxaban, eribaxaban or fondaparinux;or Factor IIa activity selected from dabigratran or argatroban.

In another embodiment, there is provided a method of treating,preventing, slowing the progression of, arresting, and/or reversingtissue calcification in a mammal undergoing hemodialysis, and in needthereof, the method comprising administering to the mammal at least 2 mgof substantially pure compound as disclosed herein per day, thereby toprevent, slow the progression, arrest, and/or reverse tissuecalcification, wherein the disclosed compound is administered in apharmaceutical composition. In another aspect, the mammal has diabetes.

Vitamin K Metabolism: Development of vascular and soft tissuecalcification following the failure to regenerate reduced forms ofvitamin K: Vitamin K is an essential enzymatic co-factor that isrequired for posttranslational modifications of vitamin K dependent(VKD) proteins. While there are numerous VKD proteins many areclinically relevant to ESRD patients. They include central coagulationfactors such as factors II VII IX and X as well as intercellular matrixproteins including Matrix GLA-1 and Osteocalcin. Under normalconditions, vitamin K is reduced to vitamin K hydroquinone (KH₂) by theenzyme NADPH oxidase. It is only the reduced form of vitamin K that isable to function as a co-factor for gamma glutamate carboxylase (GGCX)which catalyzes the carboxylation of vitamin K dependent proteins.Warfarin blocks the generation of vitamin K hydroquinone by acting as areductive sink. The enzymatic carboxylation of glutamate residuesresults in further oxidation of vitamin KH2 to 2-3 epoxide vitamin K(FIG. 2). The final step of the vitamin k cycle requires the enzymaticoxidation of vitamin K 2-3 epoxide back to its native structure. Thisstep is catalyzed by vitamin K oxidative reductase (VKOR) and is acomponent of the vitamin K cycle that is also blocked by the oxidativeeffects of Warfarin. The observation that Warfarin blocks both thegeneration of vitamin K hydroxyquinone (KH2) as well as the regenerationof Vitamin K2 2-3 epoxide helps to explains why the incidence ofcalciphylaxis and other forms of dystrophic calcification is higheramong patients receiving Warfarin therapy.

In one variation, the supplementation of the disclosed compounds andcompositions reduces the risk for vascular and soft tissue calcificationby increasing the formation of primary calciprotein particles (CPP)composed of Fetuin A and Carboxylated Matrix GLA-1 Proteins. Undernormal physiologic conditions plasma calcium and phosphateconcentrations are near supersaturation and thus would be expected toprecipitate in blood vessels and soft tissue as crystallinehydroxyapatite. The observation that this process does not occursuggests the presence of potent chemical and biologic means for blockingpathologic calcification. Recent studies have shown that circulatingcalcium phosphate crystals are complexed with two calcificationinhibiting proteins to form primary calciprotein particles (CPPs). Theseprotein-mineral complexes are composed of primarily of Fetuin A; a liverderived protein that has been shown to prevent vascular calcification. Asecond protein in lower quantities is Matrix Gla-1 protein that alsofunctions to prevent pathologic calcification. Matrix Gla-1 is a vitaminK dependent protein and early work by Price et. al and others have shownthat formation of the Fetuin-Matrix Gla-1 mineral nanoparticles (primarycalciproteins CPP) is dependent upon the gamma carboxylation of MatrixGla-1. Pre-clinical studies suggest that the calciprotein systemfunctions as an alternative means for preventing pathologiccalcification when humoral lines of defense such as pyrophosphate,magnesium and albumin are overwhelmed. The “absorption” ofcalcium-phosphate crystals by primary CCPs occurs in a coordinated andtime-dependent process.

The time to 50% saturation (T₅₀) of primary CCPs is an accurate andhighly sensitive means for determining the capacity of plasma to “sink”or “absorb” excess calcium phosphate crystals. Patients with a short T₅₀times suggests a reduced capacity to absorb calcium phosphate crystalswhereas patients with prolonged T₅₀ times are consistent with highcapacities. Recent clinical studies have validated the T₅₀ test andconfirmed that low T₅₀ times are associated with increased myocardialinfarctions, heart failure and all-cause mortality. Thus, any clinicalintervention that can increases the synthesis of circulating primaryCCPs will improve the capacity to prevent pathologic calcification. Itis noted that because patients with CKD and ESRD exhibits reduced levelsof carboxylated Matrix Gla-1 protein and that this process is essentialfor the formation of primary CPP. Accordingly, supplementation oradministration of the disclosed compounds and compositions in CKD orESRD patients will reduce the risk for pathologic calcification andprevent the development of vascular and soft tissue calcification.

Supplementation or administration of the disclosed compounds orcompositions may prevent or slow the development of soft tissue andvascular calcification in dermal tissues by restoring production ofCarboxylated Matrix Gla-1 and GAS-6.

The regeneration of Vitamin K involves two key enzymes: vitamin K 2-3epoxide oxidative reductase (VKOR) and NAD(P)H: quinone oxidoreductase(NQO1). As shown in the figure, VKOR reduces 2-3 Vitamin K epoxide tovitamin K quinone while NADPH reduces Vitamin K quinone to itshydroxyquinone form (KH2). Recent studies have shown that VKOR has twodistinct isoforms exist (VKORC-1 and VKORC1-Like-1 [VKORC1-L1]) thatdiffer in both enzymatic properties and tissue distribution. Forexample, Westhofen et. al has shown that compared to VKORC1, VKOCR-L1has a 3-fold lower affinity for 2-3 epoxide vitamin K. Subsequent worksupported the hypothesis that VKOR-L1 is a specialized isoform thatprotects against oxidant injury through the regeneration of vitamin K.When cultured HEK 293T cells were incubated with H₂O₂, VKOR-L1expression was increased and evidence of membrane oxidant injury wasreduced. The clinical observation that calciphylaxis and vitaminK-dependent vascular calcification are more common in the dermis raisesthe question of whether there is differential expression of VKOR enzymesin the skin. To address this question, Casper et. al determined mRNAexpression of key enzymes involved in regeneration of vitamin K. Asshown in FIGS. 3 and 4, skin exhibited the lowest level of VKOR-C1 thanany other tissue. Moreover, expression of NADPH in the dermis was belowthe level of detection. These observations suggest that any condition orprocedure (i.e. hemodialysis) that blocks re-constitution of vitamin Kpredisposes that tissue to pathologic calcification.

We note that the oxidative properties of uremic plasma as well as theoxidative effects of dialysis itself results in a “metabolic block” andan accumulation of 2-3 epoxide vitamin K and a reduction in theintracellular levels of vitamin K2. The “down-stream” effects of thisblockade includes the inability to gamma carboxylate key proteinsinvolved in preventing soft tissue and vascular calcification. Wefurther note that the oxidative effects of hemodialysis exacerbates thiseffect which may explain in part the predilection of ESRD patients todevelop calciphylaxis and vascular calcification.

The relationship between vitamin K and circulating vitamin K dependentproteins in CKD-ESRD Patients: It is widely recognized that despitedietary deficiencies, vitamin K levels among ESRD patients may not bereduced. For example, Holder et. al studied 172 stable dialysis patientsand found that only 6% of patients exhibited a clinically significantdeficiency in vitamin K. However, when patients were examined for thelevel of carboxylated osteocalcin, a full 60% of patients has reducedlevels. To confirm that was a general effect of reduced vitamin Kactivity, the authors also measured PIVKA-II; another vitamin Kdependent protein. Indeed, a full 90% of both CKD and ESRD patients werefound to have reduced levels of carboxylated prothrombin. In a similarstudy, Pilkey et. al measured the vitamin K1 levels in 142 ESRD patientsand found that the majority of patients had adequate vitamin K storesbut 93% of patients had uncarboxylated osteocalcin levels that weregreater than 20% of total levels. It is noted that there was nocorrelation between total vitamin K1 and the levels of circulating ofuncarboxylated osteocalcin. This unexpected finding is consistent withthe hypothesis that in uremic patients, total vitamin K levels can benormal while generation of reduced forms are blocked by the oxidativeproperties of uremia.

In one variation, the supplementation or administration of the disclosedcompounds and compositions will reverse hemodialysis induced inhibitionof vitamin K dependent proteins through normalization of functionalreduced forms of vitamin K. The observation that oxidant conditions candisrupt the vitamin K cycle suggests that the oxidant load generatedduring hemodialysis could contribute to the high rates of vascular andsoft tissue calcification observed within the ESRD population. Work byHimmelfarb et. al and others have confirmed that the simply delivery ofhemodialysis can lead to the oxidation of numerous tissue proteins. Forexample, hydroxyl amino acid side chains be oxidized to oxidized tocarbonyl groups. In a study of CKD and ESRD patients, Himmelfarb et. aldemonstrated using carbonyl side chain oxidation as a measure of globaloxidant burden, Himmelfarb et. al demonstrated that both CKD and ESRDpatients exhibit a higher percentage (15-fold) (See FIG. 5) of carbonylproteins compared to normal controls. The percentage of carbonylproteins was even higher among patients receiving dialysis demonstratingthat not only does dialysis reduce oxidant burden, it appears tocontribute to it. As shown in FIG. 5, patients with uremia were found tohave up to 15-fold higher levels of carbonylated proteins. Accordingly,the oxidative load generated by the delivery of hemodialysis leads tooxidation of the function vitamin K hydroquinone (KH2) to thenon-functional native vitamin. The oxidation of KH2 by hemodialysisblock its ability to function as a co-factor for GGCX which down-streamleads to reduced gamma carboxylation of vitamin K dependent proteins.

To confirm that uremia and hemodialysis disrupts the vitamin K cycle,the ratio of vitamin K quinone to 2-3 epoxide vitamin K and vitamin Khydroxyquinone (KH2) may be determined in patients with normal renalfunction, CKD (Stage IV & V) and ESRD patients. To determine whether thevery process of hemodialysis further disrupts the vitamin K cycle, wecan measure the levels of oxidized vitamin K in immediately prior tohemodialysis, then at mid-dialysis (2 hrs) and 30 minutes post dialysis.Previous studies examining the interactions between Warfarin and vitaminK metabolism have shown that 2-3 Epoxide Vitamin K are readily measured.Compared to controls, patients with CKD and ESRD will have higher levelsof 2-3 epoxide vitamin K and lower levels of vitamin hydroquinone (KH2).To determine whether a loss of reduced forms of Vitamin K (KH2) leads toa reduction in the carboxylation of vitamin K dependent proteins, we canmeasure the levels of the following biomarkers in control, CKD (Stage IVand V) and ESRD (Pre-Post hemodialysis). Matrix GLA-1 protein; GrowthArrest Specific Gene 6 (Gas-6) proteins; PIVAK-II protein; Osteocalcin;Protein C; Protein S; Fetuin A; and Osteoprotegerin (Dialysis PlasmaLevels: 6.7±2.2 pmole/L. We extend these studies by including patientsreceiving stable 3×/week hemodialysis. The levels of carboxylated anduncarboxylated vitamin K dependent proteins in pre-dialysis serum ma becompared levels obtained at hour 2 and the end of a dialysis session.The oxidative effects of dialysis itself will lead to a reduction in thelevel of carboxylated Vitamin K dependent proteins.

In one variation, the supplementation with the disclosed compounds andcompositions in ESRD patients with Calcific Uremic Arteriolopathy(Calciphylaxis) will reduce the time of wound healing by preventingcalcification of new blood vessels and restoring blood flow: SkinBiopsies: To confirm that supplementation of the disclosed compounds andcompositions prevents the development of small vessel calcification anddermal ischemia, we may identify patients with calciphylaxis confirmedby dermal skin biopsy and randomize patients to treatment withmenaquinone-7 or placebo. Clinical Endpoints may include thefollowing: 1) Time to Wound Vacuum therapy withdrawal and 2) time forwound healing defined as the time needed for a 50% reduction incollective the surface area of all calciphylaxis wounds.

Histopathologic Endpoints: Comparison of Diagnostic dermal biopsy withProtocol repeat dermal biopsy after 12 weeks of Menaquinone-7 therapy.Change in the level of interstitial calcium deposition defined as thechange in Von Kossa staining, which may be be quantified by digitalimage color analysis. We may use dermal biopsies to validate thebiomarkers at the tissue level. This enable the confirmation of thepreventive properties of EPN-701 on early vascular calcification. Thevalidation of these biomarkers at the tissue will also enable cliniciansto utilize the biomarkers as means to track clinical responsiveness.Calcification of microvasculature precedes development of CUA lesions.The level of calcification will be quantified by Von Kossa calciumstaining in the peripheral tissue and normalized as calcium content perunit area. We may use the Von Kossa as a means of confirming thepreventive properties of EPN-701 on the development of vascularcalcification.

In one variation, the supplementation of the disclosed compounds andcompositions in ESRD patients with Calcific Uremic Arteriolopathy(Calciphylaxis) will reduce the time of wound healing by normalizingcarboxy Protein C levels in the dermis and preventing primary thrombosisof dermal blood vessels. Accordingly, in one variation, thesupplementation or administration of the disclosed compounds orcompositions in diabetic patients will prevent the development ofvascular dementia by preventing calcification and development of smallvessel vasculopathy.

In yet another embodiment, there is provided a fortified food or drinkformulation comprising adding to the food or drink a compositioncomprising a compound of any one of the above compounds, or a mixturethereof.

Also included in the above embodiments, aspects and variations are saltsof amino acids such as arginate and the like, gluconate, andgalacturonate. Some of the compounds of the invention may form innersalts or zwitterions. Certain of the compounds of the present inventioncan exist in unsolvated forms as well as solvated forms, includinghydrated forms, and are intended to be within the scope of the presentinvention. Also provided are pharmaceutical compositions comprisingpharmaceutically acceptable excipients and a therapeutically effectiveamount of at least one compound of this invention.

Pharmaceutical compositions of the compounds of this invention, orderivatives thereof, may be formulated as solutions or lyophilizedpowders for parenteral administration. Powders may be reconstituted byaddition of a suitable diluent or other pharmaceutically acceptablecarrier prior to use. The liquid formulation is generally a buffered,isotonic, aqueous solution. Examples of suitable diluents are normalisotonic saline solution, 5% dextrose in water or buffered sodium orammonium acetate solution. Such formulations are especially suitable forparenteral administration but may also be used for oral administration.Excipients, such as polyvinylpyrrolidinone, gelatin, hydroxycellulose,acacia, polyethylene glycol, mannitol, sodium chloride, or sodiumcitrate, may also be added. Alternatively, these compounds may beencapsulated, tableted, or prepared in an emulsion or syrup for oraladministration. Pharmaceutically acceptable solid or liquid carriers maybe added to enhance or stabilize the composition, or to facilitatepreparation of the composition. Liquid carriers include syrup, peanutoil, olive oil, glycerin, saline, alcohols, or water. Solid carriersinclude starch, lactose, calcium sulfate, dihydrate, terra alba,magnesium stearate or stearic acid, talc, pectin, acacia, agar, orgelatin. The carrier may also include a sustained release material suchas glyceryl monostearate or glyceryl distearate, alone or with a wax.The amount of solid carrier varies but, preferably, will be betweenabout 20 mg to about 1 g per dosage unit. The pharmaceuticalpreparations are made following the conventional techniques of pharmacyinvolving milling, mixing, granulation, and compressing, when necessary,for tablet forms; or milling, mixing, and filling for hard gelatincapsule forms. When a liquid carrier is used, the preparation will be inthe form of a syrup, elixir, emulsion, or an aqueous or non-aqueoussuspension. Such a liquid formulation may be administered directly p.o.or filled into a soft gelatin capsule. Suitable formulations for each ofthese methods of administration may be found in, for example, Remington:The Science and Practice of Pharmacy, A. Gennaro, ed., 20th edition,Lippincott, Williams & Wilkins, Philadelphia, Pa.

As disclosed herein, the disclosed compounds and compositions mayinclude a solubility enhancer or solubilizer selected from oleic acid,Kolliphor® EL (polyoxyl castor oil or Cremophor EL), Vitamin E TPGS(D-a-tocopherol polyethylene glycol-1000 succinate), Maisine® CC(glyceryl monolinoleate), Gelucire® 44/14 (lauroyl polyoxyl-32glycerides), Miglyol® 812N (esters of saturated coconut and palm kerneloil-derived caprylic fatty acids and glycerin), Plurol® Oleique(Polyglyceryl-6 Dioleate), Lauroglycol™ 90 (propylene glycol monolaurate(type II), Labrasol® (Caprylocaproyl polyoxyl-8 glycerides), Kolliphor®EL (polyoxyl castor oil), Captisol® (SBE-beta-cyclodextrin), Encapsin™HPB (hydroxypropylbeta-cyclodextrin), Peceol™ (glycerol/glycerylmonooleate (type 40)), sodium deoxycholate, deoxycholic acid, Labrafil®M2125CS (linoleoyl Polyoxyl-6 glycerides) and medium-chain mono- anddiglycerides.

In one variation, there is provided the compounds disclosed herein, or apharmaceutically acceptable salt thereof, optionally in the form of asingle stereoisomer or mixture of stereoisomers thereof; andcompositions comprising the compounds.

In addition to the exemplary embodiments, aspects and variationsdescribed above, further embodiments, aspects and variations will becomeapparent by reference to the drawings and figures and by examination ofthe following descriptions.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless specifically noted otherwise herein, the definitions of the termsused are standard definitions used in the art of organic synthesis andpharmaceutical sciences. Exemplary embodiments, aspects and variationsare illustratived in the figures and drawings, and it is intended thatthe embodiments, aspects and variations, and the figures and drawingsdisclosed herein are to be considered illustrative and not limiting.

As used herein, a “PEG” group is a polyethylene glycol compound knownand commercially available in the art. PEG is usually a mixture ofoligomers characterized by an average molecular weight. In one example,the PEG has an average molecular weight from about 200 to about 5000. Inanother aspect, PEG has an average molecular weight from about 500 toabout 1500. In another aspect, PEG has an average molecular weight fromabout 500 to about 800 or about 900 to about 1200. In one example, thePEG is PEG-600 or is PEG-750. Both linear and branched PEG moieties canbe used in the present application. In one aspect, PEG has between 1000and 5000 subunits. In one aspect, the PEG is PEG 1000. In anotheraspect, PEG has between 100 and 500 subunits. In yet another aspect, PEGhas between 10 and 50 subunits. In one aspect, PEG has between 1 and 25subunits. In another aspect, PEG has between 15 and 25 subunits. PEG hasbetween 5 and 100 subunits. In another aspect, PEG has between 1 and 500subunits.

Similarly, an “MPEG”, “M-PEG” or “m-PEG” group is a polyoxyethanylmoiety (PEG) capped with a methyl group (methoxypolyoxyethanyl or mPEG).Accordingly, a number followed by the abbreviation “Me” (e.g., −1000Me)indicates that the PEG is capped with a methyl group, rather than ahydroxyl group, —OH.

“Pharmaceutically acceptable salts” means salt compositions that isgenerally considered to have the desired pharmacological activity, isconsidered to be safe, non-toxic and is acceptable for veterinary andhuman pharmaceutical applications. Such salts include acid additionsalts formed with inorganic acids such as hydrochloric acid, hydrobromicacid, sulfuric acid, phosphoric acid, and the like; or with organicacids such as acetic acid, propionic acid, hexanoic acid, malonic acid,succinic acid, malic acid, citric acid, gluconic acid, salicylic acidand the like.

“Therapeutically effective amount” means an amount of a compound or drugthat elicits any of the biological effects listed in the specification.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a representation of a chromatogram of menaquinone-7 and itsregioisomer shown with a ratio of 3:1, as determined by ¹H NMR.

FIG. 2 is a scheme showing the uremia and dialysis induced oxidation ofKH2 functional carboxylation of vitamin K dependent proteins.

FIG. 3 is graph showing the VKORC1 in arbitrary units and specifictissues.

FIG. 4 is a graph showing the NADPH in arbitrary units and specifictissues.

FIG. 5 is a graph showing CKD and ESRD patients exhibit a higherpercentage of carbonyl proteins compared to normal controls.

EXPERIMENTAL

The following procedures may be employed for the preparation of thecompounds of the present invention. The starting materials and reagentsused in preparing these compounds are either available from commercialsuppliers such as the Aldrich Chemical Company (Milwaukee, Wis.), Bachem(Torrance, Calif.), Sigma (St. Louis, Mo.), or are prepared by methodswell known to a person of ordinary skill in the art, followingprocedures described in such references as Fieser and Fieser's Reagentsfor Organic Synthesis, vols. 1-17, John Wiley and Sons, New York, N.Y.,1991; Rodd's Chemistry of Carbon Compounds, vols. 1-5 and supps.,Elsevier Science Publishers, 1989; Organic Reactions, vols. 1-40, JohnWiley and Sons, New York, N.Y., 1991; March J.: Advanced OrganicChemistry, 4th ed., John Wiley and Sons, New York, N.Y.; and Larock:Comprehensive Organic Transformations, VCH Publishers, New York, 1989.

In some cases, protective groups may be introduced and finally removed.Suitable protective groups for amino, hydroxy, and carboxy groups aredescribed in Greene et al., Protective Groups in Organic Synthesis,Second Edition, John Wiley and Sons, New York, 1991. Standard organicchemical reactions can be achieved by using a number of differentreagents, for examples, as described in Larock: Comprehensive OrganicTransformations, VCH Publishers, New York, 1989.

Preparation of Menaquinol-7:

Preparation of Menaquinol-7: Use of a Farnesylfarnesol-Derived Alkyneand its Subsequent Ni-Catalyzed Coupling/Reduction Reaction:

Preparation of farnesylfarnesol-derived alkyne: As described for thefarnesol-derived alkyne synthesis, farnesylfarnesol (10 g) was convertedto the corresponding chloride using PCl₃/DMF conditions. Crude chloride(10.4 g made) was obtained in 100% yield and it was sufficiently pure by¹H NMR. The crude chloride (8.9 g) was treated with dilithiopropyne andthe resulting crude alkyne was purified on a Biotage chromatographyinstrument. The alkyne was obtained as a colorless oil, 6.2 g in 69%isolated yield. Q-NMR analysis indicated that the purity of the alkyneas 94.0 wt %. This product was used as it is for the next step withoutfurther purifications (no plug filtration nor distillation). Someresidual alkyne was retained in the column and was flashed out with astronger solvent (10% EtOAc/hexane) to afford 1.6 g (18%) of additionalalkyne product as a yellow oil.

Ni-catalyzed coupling: Me₃Al (2 M in toluene, 1.5 mL, 3.0 mmol, 1.5equiv) was added to Cp₂ZrCl₂ (29 mg, 0.10 mmol, 5 mol %) at 0° C. Tothis solution was added alkyne (898 mg, 2 mmol, 1 equiv) at 0° C. Afterstirring at 0° C. for 1 h, TLC indicated most of the alkyne wasconsumed. GC/MS assay was also used to monitor this conversion process.The mixture was gently vacuumed using high vacuum pump at ambienttemperature to remove excess Me₃Al and some toluene. When the mixturebecame viscous, vacuum was stopped, leaving some residual toluene. Ifall of the toluene was removed by vacuum, an exothermic reaction toabout 35° C. was observed. The residue was cooled to—20° C. and THF (2mL) was added to the mixture. To this solution was added a THF (2 mL)solution of naphthoquinone (440 mg, 2.00 mmol, 1 equiv, lot#AP56079013-006-01). The syringe was rinsed with additional THF (1 mL)and it was added to the mixture. In a separate flask, NiCl₂(PPh₃)₂ (39mg, 0.06 mmol, 3 mol %) was suspended in THF (1 mL). To this was addedn-BuLi (2.1 M in hexane, 57 μL, 0.12 mmol, 6 mol %) and the resultinglight yellow solution was stirred for 1 min before it was transferred tothe above vinylalane and naphthoquinone mixture at −20° C. The resultingdark mixture was stirred at −20° C. for 3 h. The reaction was monitoredby TLC where no change was observed after being stirred for 1 h at −20°C. The mixture was carefully quenched by the addition of cold water and0.2 N HCl solution and then extracted with MTBE. The crude oil (1.1 g)was monitored by ¹H NMR and it indicated that the ratio of desiredisomer and the regioisomer was 96:4. Crude material was purified viaBiotage chromatography to give 840 mg of menaquinone-7 in 65% isolatedyield along with recovered naphthoquinone (150 mg, 34%). The product wasisolated by chromatography and the ratio of the isomers was 96:4 by ¹HNMR.

Synthesis of MK-7 Using Reduced Amounts of Me₃Al:

Me₃Al (2 M in toluene, 1.2 mL, 2.4 mmol, 1.2 equiv) was added toCp₂ZrCl₂ (29 mg, 0.10 mmol, 5 mol %) at 0° C. To this solution was addedthe alkyne (898 mg, 2 mmol, 1 equiv) at 0° C. After stirring at 0° C.for 1 h, TLC indicated most of the alkyne was consumed. GC/MS assay wasused to monitor the reaction progress. The mixture was gently placedunder vacuum using high vacuum pump at ambient temperature in order toremove excess Me₃Al and some toluene. When the mixture became viscous,vacuum was stopped, leaving some residual toluene. (It was noted that ifsubstantially all of the toluene was removed by vacuum, an exothermicreaction to about 35° C. was observed). The residue was cooled to −20°C. and THF (2 mL) was added to the mixture. To this solution was added aTHF (2 mL) solution of naphthoquinone (440 mg, 2.0 mmol, 1 equiv, lot#AP56079013-006-01). The syringe was rinsed with additional THF (1 mL)and it was added to the mixture. In a separate flask, NiCl₂(PPh₃)₂ (39mg, 0.06 mmol, 3 mol %) was suspended in THF (1 mL). To this was addedn-BuLi (2.1 M in hexane, 57 μL, 0.12 mmol, 6 mol %) and the resultinglight yellow solution was stirred for 1 min before it was transferred tothe above vinylalane and naphthoquinone mixture at −20° C. The resultingdark mixture was stirred at −20° C. for 1 h. ¹H NMR of the crude mixtureindicates that the ratio of MK-7 and the regioisomer was 93:7. ¹H NMRalso indicated that the formation of terminal olefin was less comparedto the crude mixture that used 1.5 equiv of Me₃Al. It was purified byBiotage chromatography to give 0.97 g of pure MK-7 in 75% isolatedyield. 52 mg of unreacted naphthoquinone (12% yield) was recovered. ¹HNMR indicated that the ratio of MK-7 and regioisomer was unchanged.

Synthesis of MK-7 with Reduced Me₃Al:

Me₃Al (2 M in toluene, 1.2 mL, 2.4 mmol, 1.2 equiv) was added toCp₂ZrCl₂ (29 mg, 0.10 mmol, 5 mol %) at 0° C. To this solution was addedalkyne (900 mg, 2 mmol, 1 equiv) at 0° C. After stirring at 0° C. for 1h, TLC indicated most of the alkyne was consumed. GC/MS assay was alsoused to monitor the reaction progress.

The mixture was cooled to −20° C. To this was added THF (2 mL) and a THF(2 mL) solution of naphthoquinone (440 mg, 2.00 mmol, 1 equiv, lot#AP56079013-006-01). The syringe was rinsed with additional THF (1 mL)and it was added to the mixture. In a separate flask, Ni(0) catalyst wasprepared as above, and it was transferred to above vinylalane andnaphthoquinone mixture at −20° C. The resulting dark mixture was stirredat −20° C. for 1 h. ¹H NMR analysis of the crude mixture indicated thatthe coupling reaction was nearly completed and the ratio of MK-7 and theregioisomer was 93:7. Comparing all the crude ¹H NMR overlaid spectra inthe above descriptions, this reaction provided the cleanest conversion.The mixture was purified on Biotage chromatography to give 1.02 g ofpure MK-7 in 78% isolated yield, with a recovery of 64 mg (15%) ofunreacted naphthoquinone. This process demonstrated that the removal ofMe₃Al and toluene from the carboalumination mixture prior to thesubsequent Negishi coupling reaction is not necessary.

Recrystallization of Menaquinone-7:

Chromatographed material (0.98 g, ratio of menaquinone-7 and theregioisomer was 93:7) was recrystallized using various solvents(toluene, dichloromethane, hexanes, heptanes, THF, methanol, ethanol,propanol, iso-propanol, MTBE, MEK, DMF and their various mixtures ofbinary and ternary solvent mixtures) in different ratios did not providea significant improvement of the regioisomeric ratio. However, thechromatographed menaquinone-7 was recrystallized from EtOAc/EtOH (1:5)provided 0.66 g (67%) of clean menaquinone-7. HPLC indicates that theratio of MK-7 and regioisomer was 99.8:0.2. Mother liquor wasconcentrated to give 0.26 g (26%) of oil and regioisomer was enriched,showing the effectiveness of the crystallization solvent mixture tooptimize the isolation of the desired isomer.

Large Scale Reactions:

The present reaction was performed using 1.2 equiv of Me₃Al with novacuuming before the coupling reaction. Farnesylfarnesol (50 g) waschlorinated using the standard method noted above, and a modified workupprocedure was used. PCl₃ (7.2 mL, 82 mmol, 0.7 equiv) was carefullyadded to DMF (240 mL) at 10° C. and vigorously stirred for 30 min. Tothis was added a DMF (30 mL) solution of farnesylfarnesol (50 g, 117mmol) using an addition funnel. The addition funnel was rinsed withadditional DMF (30 mL) and the DMF rinse was added to the mixture. Theresulting orange suspension was stirred for 1 h at 10° C. and stirredfor 1 h at ambient temperature. Because the DMF solution offarnesylfarnesol was a very thick and viscous solution, the preparationof the farnesylfarnesol solution may also be prepared in n-heptane toprovide a less viscous solution for processing and transfer at a largescale.

The reaction was monitored by LCMS until the farnesylfarnesol wasconsumed. The product was extracted with n-heptane, dried over MgSO₄ andconcentrated under reduced pressure (20 mm Hg, bath temp 38° C.). Someresidual n-heptane was allowed to remain in the chloride mixture toreduce the evaporation and loss of the chloride. The mixture wasobtained in 62.5 g of clear oil. ¹H NMR indicated 49.6 g (95%) ofdesired chloride and 12.9 g of n-heptane. This mixture was used as itwas for the next reaction without further purifications.

Preparation of the Alkyne:

Dilithiopropyne was prepared in the same manner as described above.Accordingly, instead of charging all the n-BuLi solution before propynegas was introduced, half of n-BuLi was added. After excess propyne gaswas introduced, the mixture was stirred at ambient temperature to allowthe excess propyne gas to evaporate. To the resulting propyne acetylidewas added another one equivalent of n-BuLi to form dilithiopropyne.After addition of the above crude chloride (49.6 g) and the reaction wasquenched, a 1:1 mixture of desired alkyne and unreacted chloride wasobtained. It is noted that excess propyne gas remained in the THFsolution so that the preparation of dilithiopropyne was incomplete. Thechloride decomposes in GCMS and gave multiple peaks. The chloride andthe alkyne appeared at the same retention time. The material wasre-subjected to the dilithio-alkyne displacement.

Accordingly, the dilithiopropyne was prepared again, but a scale wasplaced in the hood to weigh and monitor the weight of gaseous propynewhen it was introduced to the n-BuLi solution. To the resultingdilithiopropyne was added the above mixture of alkyne and chloride inseveral portions while monitoring the reaction aliquot by ¹H NMR, untilall chloride converted to the alkyne. The resulting crude yellow oil (60g) was monitored by ¹H NMR and shown to provide an essentially puredesired alkyne and n-heptane. GCMS data indicated that, in addition tothe alkyne, the n-butyl adduct (m/z 466.84), which was derived from theexcess n-BuLi displacement of the chloride. The crude alkyne was passedthrough a gravity grade silica-gel plug and the filter cake was rinsedwith 5% MTBE/n-heptane. After evaporation of the filtrate, 56.4 g ofclear oil (theoretical yield: 49.8 g) was obtained. ¹H NMR indicated itwas a mixture of desired alkyne and n-heptane and the purity was 76 wt%. This material was used as is for the carboalumination without furtherpurification.

The reaction using the above impure alkyne was slower compared to thepreviously made, purer alkyne. This batch of alkyne required 3 to 4hours to complete compared to within 1 h with more thoroughlychromatographed and pure alkyne.

Reaction Using Distilled and Pure Farnesylfarnesol:

100 mg of the above distilled residue of farnesylfarnesol was used forthe coupling reaction using the above cited standard procedure. Thereaction was completed within 15 min. It was noted that the reason forthe slower conversion observed above was the presence of the volatiles(likely n-heptane) that were removed to provide pure farnesulfarnesol byapplying vacuum and heat, as noted above. The n-Butyl adduct did notinterfere and the presence of n-Bu adduct was not the cause of slowerconversion. Fractions were monitored by GCMS and the fractions thatcontained pure alkyne peak were collected. 6 g of n-butyl adduct and 29g of alkyne was obtained. Since the alkyne is not volatile, it wasvacuum dried at 50° C. by rotary evaporator (10-12 mmHg) for 30 min.This material was pure by GCMS, and ¹H NMR indicated a small impuritythat may be a dimer of the chloride resulting from an attachment of acarbon-carbon bond. The Q-NMR suggested that the alkyne was 68 wt %pure.

Preparation of Menaquinone-7 Using Pure Alkyne:

A 10 g reaction scale was performed using the starting alkyne containingsome unknown impurities. Accordingly, 10 g of alkyne (14.7 g of impurealkyne as is, 61 wt %, 20.0 mmol) was treated with 1.2 equiv of Me₃Al intoluene in the presence of Cp₂ZrCl₂ (5 mol %) at 0° C. GCMS indicatedthat the reaction was completed within 1 h. The subsequent couplingreaction was carried out using 1 equiv of naphthoquinone (4.4 g, 20mmol) at −20° C. for 1 h. A reaction aliquot was monitored by ¹H NMR andindicated naphthoquinone was nearly consumed. ¹H NMR and HPLC bothindicated that the ratio of menaquinone and its regioisomer was 94:6.The reaction mixture was quenched and extracted with n-heptane. Theorganic layer was passed through a silica-gel plug and the plug wasrinsed with 5% EtOAc/n-heptane. Filtrates were concentrated to givecrude oil (21.3 g) which was recrystallized from EtOAc (20 mL) and EtOH(100 mL) to provide 15.7 g of solid (theoretical yield: 14.5 g). HPLCindicated that the ratio of desired product to the regioisomer was 97:3.Q-NMR showed a purity of 72 wt %. This product contains 11.3 g (78%) ofmenaquinone-7. The solid was stirred in n-BuOH (25 mL) at 0° C. and itwas filtered. Filter cake was rinsed with cold n-BuOH and the filtercake was dried in vacuo to afforded 9 g of pure material in 62% overallisolated yield. HPLC indicated that the ratio of menaquinone andregioisomer was 99.8:0.2, with a purity of >99.6% by HPLC using anexternal standard. HPLC Conditions: Column: Thermo Acclaim C30, 250×2.1mm, 3 μm (Part #078664); Column temperature: 15° C.; Mobile phase: 2%water in methanol; Diluent: 90% IPA in THF; Detector: UV 248 nm, 234 nm;Flow rate: 0.4 mL/min; Injection volume: 4 μL Running time: 50 min.

Reduction of MK-7 Followed by Esterification of Menaquinol-7:

The menaquinol compounds and derivatives, such as menaquinol-7 compoundsand derivatives, may be prepared according to the general scheme asdescribed below. Such acylated compounds may be symmetrical, whereinboth hydroxyl groups of the menaquinol are acylated, or only one of thetwo hydroxyl groups, either the 5-position or the 8-position, areacylated, and the other remaining as the hydroxyl group of themenaquinol.

Accordingly, the menaquinone, such as MK-7, may be contacted with ametal, such as zinc, and an acid, such as acetic acid or dilute HCl, ina protic solvent, such as methanol or ethanol, for a sufficient timeunder condition to form the corresponding menaquinol intermediate. Themenaquinol may be isolated before taking the acylation reaction, or themenaquinol may be acylated in situ with an acid halide (X=Cl, Br, I) oran acylating agent such as an acid anhydride in a solvent, to form thecorresponding mono- or di-acylated menaquinol derivative. In onevariation of the method, the acid anhydride may be a symmetrical or anunsymmetrical or mixed acid anhydride, to form the corresponding mono-or diacylated menaquinol.

To a round-bottom flask is added MK-7 (0.15 g, 0.23 mmol, 1 equiv), zincpowder (0.1 g, 1.5 mmol, 6.5 equiv), and acetic acid (0.2 mL) inmethanol (1 mL). The reaction is stirred at room temperature. After thereaction is complete, the reaction is concentrated by exposure to highvacuum to remove all volatiles, and then diluted with pyridine (1 mL).To this mixture is then added the acylating agent (2.2 equiv) and themixture is allowed to stir at rt until the hydroquinone is consumed. Thereaction mixture is then diluted with hexanes and filtrated throughCelite. The solution is then washed with a 1M HCl aqueous solution (2×20mL) and then saturated aqueous Na₂CO₃ solution. The organic layer isdried over anhydrous MgSO₄, filtered and concentrated in vacuo to yieldthe diacylated product. Further purification could be accomplished viarecrystallization or column chromatography.

To a round-bottom flask was added MK-7 (0.15 g, 0.23 mmol, 1 equiv),zinc powder (0.1 g, 1.5 mmol, 6.5 equiv) and pyridine (0.8 mL, 9.9 mmol,43 equiv) in acetic anhydride (3 mL, 138 equiv). The reaction wasstirred for 0.5 h at room temperature (at t=0 h, MK-7 is poorly solubleand the mixture is yellow; after completion, the product is welldispersed and the solution is brown). The reaction was diluted withhexanes (40 mL) and filtrated through Celite. The organic layer waswashed in succession with a 1M HCl aqueous solution (2×20 mL) andsaturated Na₂CO₃ aqueous solution. The organic layer was dried overanhydrous MgSO₄, filtered and concentrated in vacuo to yield a paleyellow oil (915 mg, 91%). R_(f)=0.46 (9:1 hexanes/Et₂O). ¹H NMR (500MHz, CDCl₃) δ 7.74-7.63 (m, 2H), 7.50-7.43 (m, 2H), 5.19-5.01 (m, 7H),3.42 (s, 2H), 2.49 (s, 3H), 2.47 (s, 3H), 2.25 (s, 3H), 2.16-1.89 (m,23H), 1.79 (s, 3H), 1.69 (s, 3H), 1.61 (s, 17H), 1.59 (s, 17H), 1.55 (s,5H). ¹³C NMR (126 MHz, CDCl₃) δ 169.5, 169.1, 142.7, 142.4, 136.4,135.3, 135.0, 135.0, 135.0, 135.0, 131.3, 130.4, 127.1, 126.4, 126.4,126.3, 126.3, 124.5, 124.4, 124.4, 124.3, 124.0, 121.5, 121.3, 121.2,39.9, 39.9, 39.8, 39.7, 29.8, 27.2, 26.9, 26.8, 26.8, 26.8, 26.7, 25.8,20.8, 20.7, 17.8, 16.5, 16.2, 16.1, 16.1, 13.2.

Reduction of menaquinone: In a two neck round bottom flask fitted with acondenser, a nitrogen purge tube and a magnetic bar is added 10 mL ofmethanol and toluene mixture (70:30) and 0.93 g ammonium formatedissolved in 1 mL water. Pd-carbon (10%) 100 mg was added after stirringfor 15 min under nitrogen followed by the menaquinone (10 mmol) afterabout 30 seconds. The mixture is stirred for 4 h at room temperature.The catalyst is removed by filtration through a sintered disk undersuction and the filtrate evaporated under reduced pressure to give about2 g of crude solid product. The residue is extracted withdichloromethane and the extract may be used in the acylation stepwithout further purification or isolation.

Depending on the reaction conditions and stoichiometry of the acylatingreagent relative to the menaquinol, the formation of the mono acylatedproduct, such as 30 and 31, or the diacylated product 32, may beprepared selectively. Accordingly, an excess of the acylation reagentwill drive the reaction toward the formation of the diacylated product32; whereas the use of less than one equivalent of the acylating reagentunder the appropriate conditions will provide the mono-acylated product.

The preparation or coupling reaction with water soluble PEG groups:

The formation of MPEG derivatives of menaquinol leads to watersolubilization of the menaquinol compounds. In one variation, the MPEGderivatives may be prepared with an initial treatment with an anhydride,such as succinic anhydride, to form the ester acid derivative. The esteracid derivative may be isolated, and then treated with an MPEG compoundto form the diesters.

To a solution of poly(ethylene glycol) monomethylether-2000 (15 g, 7.5mmol) and succinic anhydride (1.5 g, 15 mmol) in tolune (7.5 mL) andEt₃N (0.53 mL, 3.75 mmol) is added at RT, and the reaction mixture isstirred at about 60° C. for abut 8 hours until the reaction wascomplete. Water (15 mL) is added to the reaction mixture and the mixtureis extracted with DCM (3×25 mL). The combined organic layers were washedwith 1 N HCl (3×50 mL) and then with brine (2×30 mL) and then dried overanhydrous Na₂SO₄. The solution is concentrated under rotoevaporation toafford the poly(ethylene glycol) monomethyl ether-2000 succinate inabout 99% yield (15.6 g) as a solid.

Preparation of activated PEGylated Succinic Acid: Poly(ethylene glycol)monoethyl ether-2000 succinate (2.1 g, 1 mmol) is dissolved in DCM (10mL) and cooled to 0° C. N-Hydroxysuccinimide (0.14 g, 1.2 mmol) and1-(3-dimethylaminopropyl)-3-ethyl carbodiimide (EDCI, 0.25 g, 1.3 mmol)is directly added in succession to the reaction mixture as solids. Theresulting solution is stirred at RT for about 12 hours. Water (15 mL) isadded to the reaction mixture and the product is extracted with DCM(3×20 mL). The combined organic layers are washed with water (3×20 mL),brine (2×20 mL), dried over anhydrous Na₂SO₄ and concentrated viarotoevaporation to provide the product (2.17 g, 99%) as an off white,waxy solid.

NaH (0.026 g, 0.65 mmol, 60% suspension in mineral oil) is added to astirred solution of menaquinol (0.6 mmol) in THF (5 mL) at 0° C. Afterthe addition, the reaction mixture is stirred at 20° C. for about 1hour. A solution of the poly(ethylene glycol) monomethyl ether-2000succinate (0.50 mmol) in THF (50 mL) is added to the mixture at 0° C.,and the reaction is stirred for 30 minutes. The mixture is stirred foranother 8 hours at RT. The reaction is cooled to 0° C. and saturatedaqueous NH₄Cl (15 mL) is added and then extracted with DCM (3×20 mL).The combined organic layers are washed with water (2×20 mL) and brine(2×15 mL), and the organics are reduced under rotoevaporation to providea yellow liquid, which is purified by flash column chromatography onsilica gel using DCM and 1:20 MeOH/DCM gradient to provide the productin 65% yield.

Preparation of Menaquinol Compounds I:

The preparation of the diesters or mono-esters of the compound of theFormula I may be performed using standard acetylation of quinones knownin the art. For example, the quinone may be treated with a symmetricalanhydride, where R^(a) and R^(b) are the same group, or with anasymmetrical anhydride, where R^(a) and R^(b) are different groups. Inone embodiment of the symmetrical or asymmetrical anhydride, the acylgroup R^(a)—C(O)— is selected from the group consisting of:

and R^(b) may be the same as R^(a) or R^(b) may be selected from —CH₃ or—CH₂CH₃; wherein P¹ and P² are each independently a protecting groupsuch as —CH₂C₆H₅, -THP (tetrahydropyranyl) or P¹ and P² together withthe oxygen to which they are attached form a cyclic acetonide, benzylacetal or p-methoxy-benzyl acetal; P³ is a hydroxyl protecting groupsuch as -THP, acetyl, benzoyl, β-methoxyethoxymethyl ether (MEM),dimethoxytrityl, methoxymethyl ether (MOM), p-methoxybenzyl ether (PMB),methylthiomethyl ether, pivaloyl (Piv) and trityl (Tr); and each R³ andR⁴ is independently H, —CH₃, —CH₂CH₃ and —CH₂C₆H₅; and the acetylationmay be performed with the addition of a base such as Cs₂CO₃, CsHCO₃,LiCO₃, Na₂CO₃, K₂CO₃, KHCO₃, NaOAc and NaHCO₃. The acylation may beperformed in a solvent such as THF, Me-THF, toluene and ethyl acetate.

In another embodiment, the preparation of the diesters or mono-esters ofmenaquinol-7 of Formula I (R¹=R²=H) may be performed using standardacetylation of quinones using an acid halide as known in the art, wherethe halide is —Cl, —Br or —I along with a base selected from the groupconsisting of Cs₂CO₃, CsHCO₃, CsOH, LiCO₃, Na₂CO₃, K₂CO₃, KHCO₃, NaOAc,NaHCO₃, or an organic amine base as disclosed herein. For example, inone embodiment of the symmetrical or unsymmetrical anhydride, the acylgroup R^(a)—C(O)— is selected from the group consisting of the aboveresidues including 15, 16, 17, 18, 20, 21, 22, 23, 24, 25, 26, 27 and28.

Preparation of a Menaquinol I (R¹ and R² are as Defined Herein):

Menaquinone-7 (VIa, 1.00 g, 1.54 mmol), the symmetrical anhydride whereR^(a) and R^(b) are both the acyl group 15 (1.67 g, 3.08 mmol, MW=544g/ml). NaOAc (0.154 g, 1.88 mmol) and Zn powder (0.35 g, 5.44 mmol) areadded together in a 50 mL RBF equipped with a stir bar and heated toabout 140° C. for about 1 hour. The resulting mixture was cooled to RTand THF (45 mL) is added. Et₂N (20 mL) is added and the resultingmixture is stirred for another 30 minutes, and then heptane (60 mL) isadded. The resulting mixture is filtered using a buchner funnel andfilter paper, and the filtered cake is washed with heptane (2×15 mL).The solvents in the combined filtrate is removed under rotoevaporationat a water bath of about 35° C. and the resulting oil is purified byflash column chromatography (heptane:EtOAc in gradient) to provide 41.9g (about 50%) yield of the menaquinol I, where R¹ and R² are both theacyl group 15.

Preparation of a Menaquinol I (R¹=R²=H):

Menaquinone-7 (VIa, 1.00 g, 1.54 mmol), the symmetrical anhydride whereR^(a) and R^(b) are both the acyl group 15 (1.67 g, 3.08 mmol, MW=544g/ml). NaOAc (0.154 g, 1.88 mmol) and Zn powder (0.35 g, 5.44 mmol) areadded together in a 50 mL RBF equipped with a stir bar and heated toabout 140° C. for about 1 hour. The resulting mixture was cooled to RTand THF (45 mL) is added. Et₂N (20 mL) is added and the resultingmixture is stirred for another 30 minutes, and then heptane (60 mL) isadded. The resulting mixture is filtered using a buchner funnel andfilter paper, and the filtered cake is washed with heptane (2×15 mL).The solvents in the combined filtrate is removed under rotoevaporationat a water bath of about 35° C. and the resulting oil is purified byflash column chromatography (heptane:EtOAc in gradient) to provide 41.9g (about 50%) yield of the menaquinol I, where R¹ and R² are both theacyl group 15.

Administration of the Compounds of the Present Application (theDisclosed Compounds) in Subjects at Risk for Development ofCalciphylaxis:

This example describes the administration of the compounds of thepresent application to subjects at risk for development ofcalciphylaxis, but who have not yet developed the characteristic skinlesions of calciphylaxis. Risk factors to be considered include, but arenot limited to, diabetes mellitus, obesity, hemodialysis, and priortreatment with warfarin (Nigwekar et al. (2016) “A NationallyRepresentative Study of Calcific Uremic Arteriolopathy Risk Factors,” J.AM. SOC. NEPHROL. 27(11):3421-9)). The administration of these compoundscan result in protection of the subjects from skin lesions and a changein certain biomarker levels indicative of the prevention of thedevelopment of calciphylaxis.

Subjects at risk of development of calciphylaxis orally receive aselected compound of the present application at 5 mg, 10 mg, 25 mg or 50mg once daily for at least 2 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months,6 months, 1 year, or indefinitely. The dosage form is a 5 mg, 10 mg or25 mg soft-gel capsule. Two 25 mg capsules are be administered oncedaily to the 50 mg dosage cohort. It should be noted that not allsubjects with elevated risk factors for calciphylaxis will develop thecharacteristic skin lesions of calciphylaxis. The intent of treatingwith the compound of the present application proactively (prior to aclinical diagnosis of calciphylaxis) is the prevention of lesionappearance. Thus, a drop in frequency of, or elimination of lesionappearances is contemplated to be a successful treatment.

Several biomarkers can be assessed to determine the efficacy of thecompound to be administered at the three dose levels. Exemplarybiomarkers include PIVKA-II; uncarboxylated and total Matrix Gla Protein(MGP); uncarboxylated, carboxylated and total osteocalcin protein;uncarboxylated, carboxylated and total Protein C, osteoprotegerin,Fetuin A and hs-CRP. Blood samples are obtained to measure thebiomarkers according to the following schedule. Blood sampling can occurduring treatment on a weekly or monthly basis. It is contemplated thatadministration of the disclosed compounds will result in (i) an increasein PIVKA-II, which is indicative of slowing the progression of,arresting, or reversing, calciphylaxis, (ii) a decrease inuncarboxylated MGP, uncarboxylated osteocalcin, and/or uncarboxylatedProtein C, which is indicative of slowing the progression of, arresting,or reversing calciphylaxis. Further, pulse wave velocity (PWV) can bemeasured to assess arterial compliance. Improved vascular compliancewill be indicative of slowing the progression of, arresting, orreversing calciphylaxis.

Administration of the Disclosed Compounds of the Application in SubjectsDiagnosed with Calciphylaxis:

This example describes the administration of the disclosed compounds tosubjects diagnosed with calciphylaxis. Typical symptoms includepresentation of characteristic painful skin lesions (Nigwekar et al.(2015) Calciphylaxis: Risk Factors, Diagnosis, and Treatment. Am. J.Kidney Dis. 66:133-46). Definitive diagnosis of calciphylaxis isachieved via skin biopsy. Further conditions need to be considered forcorrect diagnosis.

Subjects diagnosed with calciphylaxis orally receive the disclosedcompound at 5 mg, 10 mg, 25 mg or 50 mg once daily for at least 2 weeks,4 weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely.The dosage form is a 5 mg, 10 mg or 25 mg soft-gel capsule. Two 25 mgcapsules are administered once daily to the 50 mg dosage cohort.

The arrest of, or decreases in lesion size and frequency is contemplatedto be an indication of successful treatment. The administration of thedisclosed compounds according to the foregoing will result in the arrestof, or decrease in lesion size and frequency. Additionally, becausecalciphylaxis has a considerable mortality risk, increased overallsurvival time of diagnosed subjects will be an indication of treatmentsuccess. Furthermore, the administration of the disclosed compoundsaccording to the foregoing will result in an increased overall survivaltime of diagnosed subjects.

Administration of the Disclosed Compounds in Subjects with End StageRenal Disease (ESRD) to Reverse or Slow the Progression of TissueCalcification:

This example describes the administration of the disclosed compounds toa subject with ESRD and on stable hemodialysis. The administration ofthe disclosed compounds will result in a change in aortic compliance(via plethysmography), vascular calcification and certain biomarkerlevels indicative of slowing the progression of, arresting, or reversingtissue calcification.

ESRD subjects on stable hemodialysis orally receive the disclosedcompounds at 5 mg, 10 mg, 25 mg or 50 mg once daily for least 2 weeks, 4weeks, 6 weeks, 8 weeks, 3 months, 6 months, 1 year, or indefinitely.The dosage form is a 5 mg, 10 mg or 25 mg soft-gel capsule. Two 25 mgcapsules are administered once daily to the 50 mg dosage cohort.

A 50 y.o., 65 kg male patient diagnosed with the typical symptomsassociated with moderate calciphylaxis is treated with 10 mg of thecompound of the formula I wherein m is 7, R¹ the compound of the formula24, R² is H and R³ and R⁴ are both —CH₃, for a period of 8 weeks. Afterthe treatment period, the patient is admitted and evaluated. The patientwas found to have a significant change in the examined biomarker levelssuggesting about a 10% reduction in vascular calcification, and is alsoshown to have a 10% reduction in tissue calcification.

A 65 y.o., 45 kg female patient diagnosed with the typical symptomsassociated with moderate calciphylaxis is treated with 10 mg of thecompound of the formula I wherein m is 7, R¹ the compound of the formula25, R² is H and R³ and R⁴ are both —CH₃, for a period of 10 weeks. Afterthe treatment period, the patient is admitted and evaluated. The patientwas found to have a significant change in the examined biomarker levelssuggesting about a 20% reduction in vascular calcification, and is alsoshown to have a 15% reduction in tissue calcification.

A 55 y.o., 70 kg male patient diagnosed with the typical symptomsassociated with moderate calciphylaxis is treated with 20 mg of thecompound of the formula I wherein m is 7, R¹ the compound of the formula26, R² is H and R³ and R⁴ are both —CH₃, for a period of 3 months. Afterthe treatment period, the patient is admitted and evaluated. The patientwas found to have a significant change in the examined biomarker levelssuggesting about a 25% reduction in vascular calcification, and is alsoshown to have a 20% reduction in tissue calcification.

Coronary arterial calcium scores (CAC) are used to estimate the extentof calcification of thoracic arteries. A high CAC score is indicative ofcalcification, and treatment has the aim of arresting the long termincrease in CAC score, or reversing it, or slowing the rate of increase.

Aortic plethysmography also is used to measure arterial compliance,which decreases as calcification increases. Pulse wave velocity (PWV)also is measured to assess arterial compliance. The foregoing measuresare useful in estimating the utility of treatments intended to prevent,slow the progression of, arrest or reverse vascular calcification. Thesemeasurements are used pre- and post-treatment with the disclosedcompounds to assess treatment value.

Further, several biomarkers are assessed to determine the efficacy ofthe disclosed compounds at the three dose levels. Exemplary biomarkersinclude PIVKA-II; uncarboxylated and total Matrix Gla Protein (MGP);uncarboxylated, carboxylated and total osteocalcin protein;uncarboxylated, carboxylated and total Protein C, and hs-CRP. Bloodsamples are obtained to measure the biomarkers, most conveniently duringpatient visits for hemodialysis.

The administration of the disclosed compounds can result in (i) anincrease in PIVKA-II, which is indicative of slowing the progression of,arresting or reversing tissue calcification, (ii) a decrease inuncarboxylated MGP, uncarboxylated osteocalcin, and/or uncarboxylatedProtein C, which is indicative of slowing the progression of, arrestingor reversing tissue calcification, and/or (iii) a decrease in hs-CRP,which is indicative of slowing the progression of, arresting orreversing tissue calcification and/or reduced inflammation. Followingthe daily administration of 5 mg, 10 mg, 25 mg or 50 mg of the disclosedcompounds and compositions, at least one of PIVKA-II, under-carboxylatedMatrix Gla Protein (MGP), under-carboxylated osteocalcin protein, willshow a change indicative of slowing the progression of, arresting orreversing tissue calcification.

While a number of exemplary embodiments, aspects and variations havebeen provided herein, those of skill in the art will recognize certainmodifications, permutations, additions and combinations and certainsub-combinations of the embodiments, aspects and variations. It isintended that the following claims are interpreted to include all suchmodifications, permutations, additions and combinations and certainsub-combinations of the embodiments, aspects and variations are withintheir scope.

The entire disclosures of all documents cited throughout thisapplication are incorporated herein by reference.

REFERENCES

1) Rachel M. Holden et al. Vitamins K and D Status in Stages 3-5 ChronicKidney Disease; Clin J Am Soc Nephrol 5: 590-597, 2010. 2) Pilkey, R. M.MD et al. Subclinical Vitamin K Deficiency in Hemodialysis Patients Am JKidney Dis 49:432-439, 2007. 3) Westhofen P et al. Human vitamin K2,3-epoxide reductase complex subunit 1-like 1 (VKORC1L1) mediatesvitamin K-dependent intracellular antioxidant function. J Biol Chem2011; 286: 15085-94. 4) Caspers, M. et al., Two enzymes catalyze vitaminK 2,3-epoxide reductase activity in mouse: VKORC1 is highly expressed inexocrine tissues while VKORC1L1 is highly expressed in brain. ThrombosisResearch 135:977-983, 2015. 5) Himmelfarb, J. et al., Plasma proteinthiol oxidation and carbonyl formation in chronic renal failure. KidneyInternational, Vol. 58: 2571-2578 2000. 6) Price, P. A. et al.,Discovery of a High Molecular Weight Complex of Calcium, Phosphate,Fetuin, and Matrix-Carboxyglutamic Acid Protein in the Serum ofEtidronate-treated Rats. Journal Biol Chem. 277 (6): 3926-3934, 2002. 7)Pasch, A. et al. Nanoparticle-Based Test Measures Overall Propensity forCalcification in Serum J Am Soc Nephrol 23: 1744-1752, 2012. 8)Nigwekar, S. U. et al. Vitamin K-Dependent Carboxylation of Matrix GlaProtein Influences the Risk of Calciphylaxis. J Am Soc Nephrol 28:1717-1722, 2017.

What is claimed is: 1.-46. (canceled)
 47. A method of treating,preventing, slowing the progression of, arresting, and/or reversingcalciphylaxis in a mammal in need thereof, the method comprisingadministering to the mammal a therapeutically effective amount of acomposition comprising substantially pure menaquinol compound of theFormula I:

and a pharmaceutically acceptable excipient, to prevent, slow theprogression of, arrest, or reverse calciphylaxis, wherein: m is 7, 8, 9or 10; R¹ and R² are both the residue 15; R¹ and R² are both the residue16; R¹ and R² are both the residue 17; R¹ and R² are both the residue18; R¹ and R² are both the residue 20; R¹ and R² are both the residue21; R¹ and R² are both the residue 22; R¹ and R² are both the residue23; R¹ and R² are both the residue 24; R¹ and R² are both the residue25; R¹ and R² are both the residue 26; R¹ and R² are both the residue27; R¹ and R² are both the residue 28; R¹ is H and R² is the residue 15;R² is H and R¹ is the residue 15; R¹ is H and R² is the residue 16; R²is H and R¹ is the residue 16; R¹ is H and R² is the residue 17; R² is Hand R¹ is the residue 17; R¹ is H and R² is the residue 18; R² is H andR¹ is the residue 18; R¹ is H and R² is the residue 20; R² is H and R¹is the residue 20; R¹ is H and R² is the residue 21; R² is H and R¹ isthe residue 21; R¹ is H and R² is the residue 22; R² is H and R¹ is theresidue 22; R¹ is H and R² is the residue 23; R² is H and R¹ is theresidue 23; R¹ is H and R² is the residue 24; R² is H and R¹ is theresidue 24; R¹ is H and R² is the residue 25; R² is H and R¹ is theresidue 25; R¹ is H and R² is the residue 26; R² is H and R¹ is theresidue 26; R¹ is H and R² is the residue 27; R² is H and R¹ is theresidue 27; and R¹ is H and R² is the residue 28; R² is H and R¹ is theresidue
 28.


48. The method of claim 47, wherein the method comprises administeringthe menaquinol compound of the Formula I, wherein: m is 7, 8, 9 or 10;R¹ and R² are both the residue 15; R¹ and R² are both the residue 16; R¹and R² are both the residue 17; R¹ and R² are both the residue 18; R¹and R² are both the residue 20; R¹ and R² are both the residue 21; R¹and R² are both the residue 22; R¹ and R² are both the residue 23; R¹and R² are both the residue 24; R¹ and R² are both the residue 25; R¹and R² are both the residue 26; R¹ and R² are both the residue 27; andR¹ and R² are both the residue
 28. 49. The method of claim 47, whereinthe method comprises administering the menaquinol compound of theFormula I, wherein: m is 7, 8, 9 or 10; R¹ is H and R² is the residue15; R² is H and R¹ is the residue 15; R¹ is H and R² is the residue 16;R² is H and R¹ is the residue 16; R¹ is H and R² is the residue 17; R²is H and R¹ is the residue 17; R¹ is H and R² is the residue 18; R² is Hand R¹ is the residue 18; R¹ is H and R² is the residue 20; R² is H andR¹ is the residue 20; R¹ is H and R² is the residue 21; R² is H and R¹is the residue 21; R¹ is H and R² is the residue 22; R² is H and R¹ isthe residue 22; R¹ is H and R² is the residue 23; R² is H and R¹ is theresidue 23; R¹ is H and R² is the residue 24; R² is H and R¹ is theresidue 24; R¹ is H and R² is the residue 25; R² is H and R¹ is theresidue 25; R¹ is H and R² is the residue 26; R² is H and R¹ is theresidue 26; R¹ is H and R² is the residue 27; R² is H and R¹ is theresidue 27; and R¹ is H and R² is the residue 28; R² is H and R¹ is theresidue
 28.


50. The method of claim 47, wherein the method comprises administeringthe menaquinol compound of the Formula I, wherein: m is 7, 8, 9 or 10;R¹ is H and R² is the acyl residue of caffeic acid 20; or R² is H and R¹is the acyl residue of caffeic acid 20


51. The method of claim 47, wherein the method comprises administeringthe menaquinol compound of the Formula I, wherein: m is 7, 8, 9 or 10;R¹ is H and R² is the acyl residue of ferulic acid 21; R² is H and R¹ isthe acyl residue of ferulic acid 21;


52. The method of claim 47, wherein the method comprises administeringthe menaquinol compound of the Formula I, wherein: m is 7, 8, 9 or 10;R¹ is H and R² is the acyl residue of chlorogenic acid 22; R² is H andR¹ is the acyl residue of chlorogenic acid 22;


53. The method of claim 47, wherein the method comprises administeringthe menaquinol compound of the Formula I, wherein: m is 7, 8, 9 or 10;R¹ is H and R² is the acyl residue of quinic acid 23; R² is H and R¹ isthe acyl residue of quinic acid 23;


54. The method of claim 47, wherein the mammal has distal calciphylaxisand/or central calciphylaxis.
 55. The method of claim 47, wherein themammal has diabetes, chronic kidney disease or end stage renal disease.56. A method of treating calciphylaxis in a mammal in need thereof, themethod comprising administering to the mammal a therapeuticallyeffective amount of a composition comprising substantially puremenaquinol compound of the Formula I:

and a pharmaceutically acceptable excipient, to treat calciphylaxis,wherein: m is 7, 8, 9 or 10; R¹ and R² are both the residue 15; R¹ andR² are both the residue 16; R¹ and R² are both the residue 17; R¹ and R²are both the residue 18; R¹ and R² are both the residue 20; R¹ and R²are both the residue 21; R¹ and R² are both the residue 22; R¹ and R²are both the residue 23; R¹ and R² are both the residue 24; R¹ and R²are both the residue 25; R¹ and R² are both the residue 26; R¹ and R²are both the residue 27; R¹ and R² are both the residue 28; R¹ is H andR² is the residue 15; R² is H and R¹ is the residue 15; R¹ is H and R²is the residue 16; R² is H and R¹ is the residue 16; R¹ is H and R² isthe residue 17; R² is H and R¹ is the residue 17; R¹ is H and R² is theresidue 18; R² is H and R¹ is the residue 18; R¹ is H and R² is theresidue 20; R² is H and R¹ is the residue 20; R¹ is H and R² is theresidue 21; R² is H and R¹ is the residue 21; R¹ is H and R² is theresidue 22; R² is H and R¹ is the residue 22; R¹ is H and R² is theresidue 23; R² is H and R¹ is the residue 23; R¹ is H and R² is theresidue 24; R² is H and R¹ is the residue 24; R¹ is H and R² is theresidue 25; R² is H and R¹ is the residue 25; R¹ is H and R² is theresidue 26; R² is H and R¹ is the residue 26; R¹ is H and R² is theresidue 27; R² is H and R¹ is the residue 27; and R¹ is H and R² is theresidue 28; R² is H and R¹ is the residue
 28.


57. The method of claim 56, wherein the method comprises administeringthe menaquinol compound of the Formula I, wherein: m is 7, 8, 9 or 10;R¹ and R² are both the residue 15; R¹ and R² are both the residue 16; R¹and R² are both the residue 17; R¹ and R² are both the residue 18; R¹and R² are both the residue 20; R¹ and R² are both the residue 21; R¹and R² are both the residue 22; R¹ and R² are both the residue 23; R¹and R² are both the residue 24; R¹ and R² are both the residue 25; R¹and R² are both the residue 26; R¹ and R² are both the residue 27; andR¹ and R² are both the residue
 28. 58. The method of claim 56, whereinthe method comprises administering the menaquinol compound of theFormula I, wherein: m is 7, 8, 9 or 10; R¹ is H and R² is the residue15; R² is H and R¹ is the residue 15; R¹ is H and R² is the residue 16;R² is H and R¹ is the residue 16; R¹ is H and R² is the residue 17; R²is H and R¹ is the residue 17; R¹ is H and R² is the residue 18; R² is Hand R¹ is the residue 18; R¹ is H and R² is the residue 20; R² is H andR¹ is the residue 20; R¹ is H and R² is the residue 21; R² is H and R¹is the residue 21; R¹ is H and R² is the residue 22; R² is H and R¹ isthe residue 22; R¹ is H and R² is the residue 23; R² is H and R¹ is theresidue 23; R¹ is H and R² is the residue 24; R² is H and R¹ is theresidue 24; R¹ is H and R² is the residue 25; R² is H and R¹ is theresidue 25; R¹ is H and R² is the residue 26; R² is H and R¹ is theresidue 26; R¹ is H and R² is the residue 27; R² is H and R¹ is theresidue 27; and R¹ is H and R² is the residue 28; R² is H and R¹ is theresidue
 28.


59. The method of claim 56, wherein the method comprises administeringthe menaquinol compound of the Formula I, wherein: m is 7, 8, 9 or 10;R¹ is H and R² is the acyl residue of caffeic acid 20; or R² is H and R¹is the acyl residue of caffeic acid 20


60. The method of claim 56, wherein the method comprises administeringthe menaquinol compound of the Formula I, wherein: m is 7, 8, 9 or 10;R¹ is H and R² is the acyl residue of ferulic acid 21; R² is H and R¹ isthe acyl residue of ferulic acid 21;


61. The method of claim 56, wherein the method comprises administeringthe menaquinol compound of the Formula I, wherein: m is 7, 8, 9 or 10;R¹ is H and R² is the acyl residue of chlorogenic acid 22; R² is H andR¹ is the acyl residue of chlorogenic acid 22;


62. The method of claim 56, wherein the method comprises administeringthe menaquinol compound of the Formula I, wherein: m is 7, 8, 9 or 10;R¹ is H and R² is the acyl residue of quinic acid 23; R² is H and R¹ isthe acyl residue of quinic acid 23;


63. The method of claim 56, wherein the mammal has distal calciphylaxisand/or central calciphylaxis.
 64. The method of claim 56, wherein themammal has diabetes, chronic kidney disease or end stage renal disease.65. A method of reversing calciphylaxis in a mammal in need thereof, themethod comprising administering to the mammal a therapeuticallyeffective amount of a composition comprising substantially puremenaquinol compound of the Formula I:

and a pharmaceutically acceptable excipient, to reverse calciphylaxis,wherein: m is 7, 8, 9 or 10; R¹ and R² are both the residue 15; R¹ andR² are both the residue 16; R¹ and R² are both the residue 17; R¹ and R²are both the residue 18; R¹ and R² are both the residue 20; R¹ and R²are both the residue 21; R¹ and R² are both the residue 22; R¹ and R²are both the residue 23; R¹ and R² are both the residue 24; R¹ and R²are both the residue 25; R¹ and R² are both the residue 26; R¹ and R²are both the residue 27; R¹ and R² are both the residue 28; R¹ is H andR² is the residue 15; R² is H and R¹ is the residue 15; R¹ is H and R²is the residue 16; R² is H and R¹ is the residue 16; R¹ is H and R² isthe residue 17; R² is H and R¹ is the residue 17; R¹ is H and R² is theresidue 18; R² is H and R¹ is the residue 18; R¹ is H and R² is theresidue 20; R² is H and R¹ is the residue 20; R¹ is H and R² is theresidue 21; R² is H and R¹ is the residue 21; R¹ is H and R² is theresidue 22; R² is H and R¹ is the residue 22; R¹ is H and R² is theresidue 23; R² is H and R¹ is the residue 23; R¹ is H and R² is theresidue 24; R² is H and R¹ is the residue 24; R¹ is H and R² is theresidue 25; R² is H and R¹ is the residue 25; R¹ is H and R² is theresidue 26; R² is H and R¹ is the residue 26; R¹ is H and R² is theresidue 27; R² is H and R¹ is the residue 27; and R¹ is H and R² is theresidue 28; R² is H and R¹ is the residue
 28.


66. The method of claim 65, wherein the mammal has distal calciphylaxisand/or central calciphylaxis.
 67. The method of claim 65, wherein themammal has diabetes, chronic kidney disease or end stage renal disease.